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  • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
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  • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
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  • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
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  • C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
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  • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
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  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
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  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
  • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
  • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
  • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
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  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
  • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
  • G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
  • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
  • G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
  • G01N33/57488—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites involving compounds identifable in body fluids
• • A—HUMAN NECESSITIES
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  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
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  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
  • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
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  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
  • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
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  • C12N2310/00—Structure or type of the nucleic acid
  • C12N2310/10—Type of nucleic acid
  • C12N2310/16—Aptamers

Definitions

• Antagonist to an enzyme and/or a metabolite of the kynurenine pathway Antagonist to an enzyme and/or a metabolite of the kynurenine pathway
• the present invention is related to an antagonist to an enzyme and/or a metabolite of the kynurenine pathway.
• Tumor progression implicates several factor named as "hallmarks of cancer” (Hanahan et al 2011) Among them, resistance to cell death, exaggerated proliferation, genomic instability, and tumor angio genesis are factors that promote tumor growth.
• tumor immune escape is a key factor in cancer progression, which has been neglected for long time.
• a cell becomes malignant by transformation, i.e., when it becomes different from the "self, it should be recognized and eliminated by the immune system. Accordingly, transformed cells are continuously eliminated in our organism by a functional immune system.
• a tumor cell is, in some cases, able to repress the immune response. This process is called the “immune escape” or “tumor escape” (Dunn et al 2002) and is partially controlled by the indoleamine 2,3 dioxygenase (IDOl) / tryptophan 2,3 dioxygenase (TD02) enzymes (Prendergast 2008; Opitz 2011).
• IDOl indoleamine 2,3 dioxygenase
• TD02 tryptophan 2,3 dioxygenase
• IDOl inhibition by 1 -Methyl Tryptophan (1MT) the prototypical IDOl/2 inhibitor
• chemotherapeutic drugs such as cyclophosphamide or paclitaxel
• LM-10 trans-6-Fluoro-3-[2-(lH-tetrazol-5-yl)vinyl]-lH- indole
• IDOl is overexpressed in many types of tumour with the highest frequency found in colorectal and pancreas cancers and glioblastoma (Uyttenhove et al 2003). Moreover, increasing expression of IDOl in colorectal cancer is associated with a poor prognosis (Ferdinande et al 2012). It was shown recently that TD02 is a new enzyme responsible of tryptophan catabolism in major types of cancer (Pilotte et al 2012).
• TD02 overexpression was presented to promote glioblastoma progression by acting on both the immune and the tumoral compartments (Opitz et al 2012).
• Table 1 we summarize the different expression level of IDOl and TD02 in different types of tumors with their respective clinical significance.
• a modulator of an enzyme and/or a metabolite of the kynurenine pathway is provided.
• the term "modulator of an enzyme and/or a metabolite of the kynurenine pathway” refers to a substance that affects at least one selected from the group consisting of the formation, concentration, availability, metabolism and/or effect of an enzyme and/or a metabolite of the kynurenine pathway.
• Such modulator does not necessarily have to bind to said enzyme and/or a metabolite of the kynurenine pathway. It is sufficient if said modulator exerts its effect even without binding, e.g., indirectly, e.g. by (i) affecting an enzyme which is causative for the formation of said target metabolite, (ii) affecting a co-factor which the target enzyme needs, and/or (iii) affecting the molecular target of said enzyme and/or a metabolite, in such way limiting a potential pathological effect of the latter.
• Said enzyme and/or a metabolite of the kynurenine pathway can either be a in soluble form, or attached to another moiety (e.g., bound to a membrane or the like, attached to a cofactor, or the like).
• the term "kynurenine pathway” encompasses enzymes and metabolites of said pathway with the exception of indoleamine 2,3 dioxygenase 1 and 2 (IDO 1/2) and tryptophan 2,3 dioxygenase 2 (TD02). Either of the former two enzymes catalyzes the formation of N- Formylkynurenine (NFK) from Tryptophan.
• IDO 1/2 indoleamine 2,3 dioxygenase 1 and 2
• TD02 tryptophan 2,3 dioxygenase 2
• the kynurenine pathway is a metabolic pathway leading to the production of nicotinamide adenine dinucleotide (NAD+) from the degradation of the essential amino acid tryptophan.
• NAD+ nicotinamide adenine dinucleotide
• the kynurenine pathway is involved in physiological functions such as behavior, sleep, thermo -regulation and pregnancy.
• kynurenine pathway involvement in neurotoxic mechanisms associated with several inflammatory neurological diseases. Although the pathway is activated in these disorders, kynurenine and its metabolites can play both neurotoxic and neuroprotective roles by influencing neurotransmitter functions and inflammatory pathways peripherally and within the central nervous system.
• said modulator is an antagonist to said enzyme and/or a metabolite of the kynurenine pathway.
• the term "antagonist” shall encompass all moieties that have an affinity to an enzyme and/or a metabolite of the kynurenine pathway, or a conjugate thereof, but no efficacy This means, for example, that (i) upon binding of said antagonist to the enzyme and/or metabolite no physiological function is elicited, or a dampened or altered physiological function is elicited, (ii) binding of said antagonist to the enzyme and/or metabolite inhibits the binding thereof to its physiological counterpart, or (iii) binding of said antagonist to said metabolite inhibits metabolism thereof. Binding will thus disrupt the interaction between the enzyme or metabolite, or a conjugate thereof to its physiological target, and thus inhibit its role in pathological processes.
• administering affects at least one selected from the group consisting of the formation, concentration, availability and/or effect of 3HAA (3-Hydroxy Anthranilic Acid), L-Kynurenine, Quinolinic Acid and/or Cinnabarinic Acid.
• 3HAA 3-Hydroxy Anthranilic Acid
• L-Kynurenine L-Kynurenine
• Quinolinic Acid and/or Cinnabarinic Acid.
• the term "formation of a metabolite” means metabolic synthesis thereof, e.g., in the kynurenine pathway.
• concentration of a metabolite means concentration thereof in one or more selected tissues, plasma serum levels, and the like.
• effect of a metabolite relates to any downstream function the metabolite may have. By non-restricting example such function may be, e.g., a metabolite function as well as a co-factor function or a 2 nd messenger function.
• 3HAA, L-Kynurenine, Quinolinic Acid and/or Cinnabarinic Acid seem to be key metabolites that may be used as a therapeutic target. Without being bound to theory, it is assumed that, for example, 3HAA, L-Kynurenine, Quinolinic Acid and/or Cinnabarinic Acid seem to play a key role in the immune escape and tumor growth, blocking of which may thus restore the function of the immune system against the tumor and its oncological properties.
• said enzyme of the kynurenine pathway is at least one selected from the group consisting of Kynurenine formamidase, Kynurenine aminotransferase, Kynurenine 3-hydroxylase (also called Kynurenine mono-oxygenase), Kynureninase (also called L-Kynurenine hydrolase), Kynurenine amino-transferase, and/or 3- Hydroxyanthranilic Acid oxygenase (also called 3-Hydroxanthranilate dioxygenase).
• said metabolite of the kynurenine pathway is at least one selected from the group consisting of N-Formylkynurenine, D and/or L-Kynurenine, Kynurenic acid, Quinaldic acid, Kynuramine, 3-hydroxy-L-kynurenine, 3-hydroxy-D- kynurenine, Xanthommatin, Anthranilic Acid, Xanthurenic Acid, 3-Hydroxy Anthranilic Acid, Picolinioc Acid and/or Quinolinic Acid and/or Cinnabarinic Acid.
• FIG. 20 An overview of particularly preferred enzymes and metabolites of the kynurenine pathway is shown in Fig. 20.
• the modulator and/or antagonist to an enzyme and/or a metabolite of the kynurenine pathway is an antagonist to 3HAA (3-Hydroxy Anthranilic Acid), L-Kynurenine, Quinolinic Acid and/or Cinnabarinic Acid.
• the respective metabolite can be present either be a in soluble form, or attached to another moiety (e.g., bound to a membrane or the like, attached to a co factor, or the like).
• such antagonist can inactivate the cancer-promoting effect of the metabolite, e.g., by avoiding binding thereof to an enzyme or receptor, or avoiding further metabolic degradation thereof.
• the modulator and/or antagonist to an enzyme and/or a metabolite of the kynurenine pathway is an antagonist to the enzyme Kynureninase.
• This enzyme catalyzes one step of the kynurenine pathway, namely the transformation of 3- Hydroxykynurenine to 3HAA (3-Hydroxy Anthranilic Acid). Blocking of this enzme can effectively reduce the formation and/or concentration of 3HAA (3-Hydroxy Anthranilic Acid).
• Two non-restricting examples of preferred antagonists to Kynureninase are O-methoxybenzoylalanine 2-amino-4-[3'-hydroxyphenyl]-4-hydroxybutanoic acid.
• said human or animal suffers from a neoplastic disease.
• neoplastic disease refers to an abnormal state or condition of cells or tissue characterized by rapidly proliferating cell growth or neoplasm. In a more specific meaning, the term relates to cancerous processes, e.g., tumors and/or leukemias.
• said human or animal suffers from a disease caused by over- or underabundancy of an enzyme and/or metabolite of the kynurenine pathway.
• said neoplastic disease is at least one disease characterized by tryptophan metabolism exaggeration along the kynurenine pathway. Tryptophan metabolism exaggeration is defined as a decrease in tryptophan level and/or increase of one of the kynurenine pathway metabolites.
• said neoplastic disease is at least one disease characterized by any form of activity of Indoleamine 2,3-dioxygenase l(IDOl) and/or Indoleamine 2,3-dioxygenase 2 (ID02) and/or Tryptophan 2,3-dioxygenase (TD02).
• Table 1 shows an non-exhaustive list of cancers characterized by overexpression of IDOl and/or TD02.
• IHC means “Immunohistochenmistry” and “Tregs” mean "Regulatory T cells”
• X means “reduces” and “ ⁇ ” means “increases”.
• the inventors have, surprisingly, conceived that a relationship between the kynurenine pathway and cancer genesis exists. This finding applies to all cancers characterized by overexpression of IDOl and/or TD02.
• said neoplastic disease is selected from the group consisting of
• said modulator and/or antagonist to an enzyme and/or a metabolite of the kynurenine pathway serves to inhibit processes, in the human or animal, related to immune escape and/or immuno editing.
• Tumor escape is a key factor in cancer progression, which has been neglected for long time.
• a cell becomes malignant by transformation, i.e., when it becomes different from the "self, it should be recognized and eliminated by the immune system. Accordingly, transformed cells are continuously eliminated in our organism by a functional immune system.
• a tumor cell is, in some cases, able to repress the immune response. This process is called the immune escape. All processes related to the immune escape are charcterized as "immunoediting".
• said modulator and/or antagonist to an enzyme and/or a metabolite of the kynurenine pathway is at least one selected from the group consisting of
• a fusion peptide comprising at least one domain capable of binding an enzyme and/or a metabolite of the kynurenine pathway
• mAb monoclonal antibody
• mAb monoclonal antibody
• the term "monoclonal antibody (mAb)” shall refer to an antibody composition having a homogenous antibody population, i.e., a homogeneous population consisting of a whole immunoglobulin, or a fragment or derivative thereof.
• mAb is selected from the group consisting of IgG, IgD, IgE, IgA and/or IgM, or a fragment or derivative thereof.
• fragment shall refer to fragments of such antibody retaining, in some cases, target binding capacities, e.g.
• IgG heavy chain consisting of VH, CHI, hinge, CH2 and CH3 regions
• IgG light chain consisting of VL and CL regions
• derivative shall refer to protein constructs being structurally different from, but still having some structural relationship to, the common antibody concept, e.g., scFv, Fab and/or F(ab) 2 , as well as bi-, tri- or higher specific antibody constructs. All these items are explained below.
• IgG, scFv, Fab and/or F(ab) 2 are antibody formats well known to the skilled person. Related enabling techniques are available from the respective textbooks.
• Fab relates to an IgG fragment comprising the antigen binding region, said fragment being composed of one constant and one variable domain from each heavy and light chain of the antibody
• F(ab) 2 relates to an IgG fragment consisting of two Fab fragments connected to one another by disulfide bonds.
• scFv relates to a single-chain variable fragment being a fusion of the variable regions of the heavy and light chains of immunoglobulins, linked together with a short linker, usually serine (S) or glycine (G). This chimeric molecule retains the specificity of the original immunoglobulin, despite removal of the constant regions and the introduction of a linker peptide.
• new antibody formats encompasses, for example bi- or trispecific antibody constructs, Diabodies, Camelid Antibodies, Domain Antibodies, bivalent homodimers with two chains consisting of scFvs, IgAs (two IgG structures joined by a J chain and a secretory component), shark antibodies, antibodies consisting of new world primate framework plus non-new world primate CDR, dimerised constructs comprising CH3+VL+VH, and antibody conjugates (e.g., antibody or fragments or derivatives linked to a toxin, a cytokine, a radioisotope or a label).
• antibody conjugates e.g., antibody or fragments or derivatives linked to a toxin, a cytokine, a radioisotope or a label.
• immunotoxins i.e., heterodimeric molecules consisting of an antibody, or a fragment thereof, and a cytotoxic, radioactive or apoptotic factor.
• immunotoxins i.e., heterodimeric molecules consisting of an antibody, or a fragment thereof, and a cytotoxic, radioactive or apoptotic factor.
• Such type of format has for example been developed by Philogen (e.g., anti-EDB human antibody L19, fused to human TNF), or Trastuzumab emtansine (T-DM1), which consists of trastuzumab linked to the cytotoxoic Mertansine (DM1).
• L-Kynurenine, 3HAA, Cinnabarinic acid are overabundant in tumor tissue, targeting these metabolites with a specific immunotoxin represents a very promising therapeutic approach of site-directed tumor therapy.
• fusion peptide or “fusion protein” proteins relates, for example, to proteins consisting of an immunoglobulin Fc portion plus a target binding moiety capable of binding an enzyme and/or a metabolite of the kynurenine pathway (so-called -cept molecules).
• antibody mimetic relates to target binding proteins, which are not related to immunoglobulins. Many of the above mentioned techniques, like phage display, are applicable for these molecules as well.
• Such antibody mimetics are for example derived from Ankyrin Repeat Proteins, C-Type Lectins, A-domain proteins of Staphylococcus aureus, Transferrins, Lipocalins, Fibronectins, Kunitz domain protease inhibitors, Ubiquitin, Cysteine knots or knottins, thioredoxin A, and so forth, and are known to the skilled person in the art from the respective literature.
• aptamer relates to nucleic Acid species, which are capable of binding to molecular targets such as small molecules, proteins, nucleic Acids, and even cells, tissues and organisms.
• Aptamers are useful in biotechno logical and therapeutic applications as they offer molecular recognition properties that rival that of the commonly used biomolecule, antibodies.
• aptamers offer advantages over antibodies or other target binders as they can be engineered completely in a test tube, are readily produced by chemical synthesis, possess desirable storage properties, and elicit little or no immunogenicity in therapeutic applications.
• Aptamers can for example be produced through repeated rounds of in vitro selection or equivalent ly, SELEX (systematic evolution of ligands by exponential enrichment) to bind
• small molecule antagonist relates to a low molecular weight organic compound, which is by definition not a polymer.
• small molecule especially within the field of pharmacology, is usually restricted to a molecule that also binds with high affinity to a biopolymer such as protein, nucleic Acid, or polysaccharide and in addition alters the activity or function of the biopolymer.
• the upper molecular weight limit for a small molecule is often set at 800 Daltons, which allows for the possibility to rapidly diffuse across cell membranes so that they can reach intracellular sites of action. In addition, this molecular weight cutoff is a necessary but insufficient condition for oral bioavailability.
• Small molecules acting as antagonists against a given target e.g., an enzyme and/or a metabolite of the kynurenine pathway
• a given target e.g., an enzyme and/or a metabolite of the kynurenine pathway
• said modulator and/or antagonist to an enzyme and/or a metabolite of the kynurenine pathway is conjugated to a given carrier.
• Said conjugation to a given carrier may serve to increase the bioavailability, the efficiency and/or the serum half- life of the modulator and/or antagonist according to the invention.
• PEGylation involves the modification of a protein, peptide, or non-peptide molecule by linking of one or more polyethylene glycol chains to it, and thus results in a prolonged serum half-life particularly of smaller protein drugs, like antibody fragments, as for example put into practice in the pegylated Fab fragment Certolizumab pegol.
• N-glycosylation sites are introduced into said protein therapeutic.
• additional N-glycosylation motifs i.e., tripeptide sequences Asn-X-Ser or Asn-X-Thr, where X can be any amino Acid (although Pro and Asp are rarely found).
• the antibody, or fragment or derivative thereof has, somewhere in its chain, the motif "Gly-X-Ser", one could substitute "Gly” by "Asn”, on order to create an additional N-glycosylation site. It is of course necessary to make sure that the said substitution does not affect important properties of the protein, like target affinity, binding by Fc gamma receptors (FcyRs) or the like.
• Increasing half-life can further be obtained by conjugating the said antagonist to an enzyme and/or a metabolite of the kynurenine pathway to a polypeptidic carrier such as Poly-L-Lysine or modified Poly-L-Lysine.
• a polypeptidic carrier such as Poly-L-Lysine or modified Poly-L-Lysine.
• This method encompasses the covalent binding of the antagonist to Poly-Lysine and/or modified Poly-L-Lysine.
• Modified Poly-L-Lysine can be obtained by adding at least one other moiety.
• Conjugating the antagonist to Poly-Lysine can be performed by using crosslinkers that react with the free amine group of the Poly-L-Lysine and reactive functions on the antagonist. Grafting the antagonist on modified poly-l-lysine can be performed by crosslinking of the reactive groupement of antagnist to new reactive functions of the Pol-L-Lysine.
• Crosslinkers can be for example glutaraldehyde (NH 2 to NH 2 ), EDC (NH 2 to COOH), SMCC (NH 2 to SH). This conjugate would be able to limit liver and kidney filtration of the therapeutic protein as well as limiting its degradation by specific enzymes and therefore increasing its efficiency.
• a combination preparation comprising at least (i) the modulator and/or antagonist to an enzyme and/or a metabolite of the kynurenine pathway according to any of the aforementioned claims and (ii) at least one more active substance selected from the group consisting of
• the term "antineoplastic agent” relates to a drug, or a combination of drugs, which have antineoplastic or anticancer effects. This applies, above all, to chemotherapeutic agents, which work by impairing mitosis, effectively targeting fast-dividing cells, or by causing cells to undergo apoptosis.
• chemotherapeutic agents which work by impairing mitosis, effectively targeting fast-dividing cells, or by causing cells to undergo apoptosis.
• the majority of chemotherapeutic drugs can be divided into alkylating agents, antimetabolites, anthracyclines, plant alkaloids, topoisomerase inhibitors, and other antitumour agents.
• Targeted drugs are a type of medication that blocks the growth of cancer cells by interfering with specific targeted molecules needed for carcinogenesis and tumor growth, rather than by simply interfering with rapidly dividing cells (e.g. with traditional chemotherapy).
• the main categories of targeted therapy are small molecules and monoclonal antibodies.
• Small molecules falling under this definition encompass, but are not limited, to Imatinib, Gefitinib, Erlotinib, Bortezomib, Bcl-2 inhibitors (e.g. Obatoclax, ABT-263, and Gossypol), PARP inhibitors (e.g. Iniparib, Olaparib), Janus kinase inhibitors, PI3K inhibitors, Apatinib, AN- 152, Doxorubicin linked to [D-Lys(6)]- LHRH, Pegaptanib, Sunitinib, Sorafenib, Tivozanib and Pazopanib.
• Bcl-2 inhibitors e.g. Obatoclax, ABT-263, and Gossypol
• PARP inhibitors e.g. Iniparib, Olaparib
• Janus kinase inhibitors e.g. Iniparib, Olaparib
• PI3K inhibitors
• Endocrine drugs are drugs that are antagonistic to hormones or hormone receptors and thus interfere with cancer types that require hormones to grow.
• Endocrine drug is Tamoxifen, which is an antagonist of the estrogen receptor in breast tissue.
• cellular therapy shall relate to cell-based therapies such as adoptive transfer of modified, or unmodified, cytotoxic lymphocytes or dendritic cells.
• tumor vaccine refers to vaccines that either a) prevent infections with cancer-causing viruses (mode of action is similar to other vaccines against viral infections), b) treat existing cancer (therapeutic cancer vaccines) or c) prevent the
• tumor vaccine of type b) or c) also called “immunotherapeutic” herein
• immunotherapeutic is to isolate proteins from cancer cells and immunize cancer patients against those proteins, in the hope of stimulating an immune reaction that would kill the cancer cells.
• Another approach to therapeutic anti-cancer vaccination is to generate the immune response in situ in the patient. This enhances the anti-tumor immune response to tumor antigens released following lytic virus replication providing an in situ, patient specific anti-tumor vaccine as a result.
• Yet another approach is to immunize the patient with a compound that play a physiological role in cancer genesis, so that the human body eliminates said compound.
• the compound is a self-antigen or self hapten, i.e., it does not provoke a strong immune response when administered to the patient. It has thus to be conjugated to a given carrier.
• the use of the modulator and/or antagonist to an enzyme and/or a metabolite of the kynurenine pathway according to any of the aforementioned claims for the treatment of a neoplastic disease is provided.
• said use is complemented, in a coordinated fashion, by the administration of at least one active substance selected from the group consisting of
• complemented, in a coordinated fashion shall refer to a coadministration, which is carried out under a given regimen. This includes synchronous administration of the different compounds as well as time-shifted administration of the different compounds (e.g., compound A is given once and compound B is given several times thereafter, or vice versa, or both compounds are given synchronously and one of the two is also given at later stages).
• said use is complemented, in a coordinated fashion, by at least one other treatment selected from the group consisting of
• a modulator and/or antagonist to an enzyme and/or a metabolite of the kynurenine pathway according to any of the aforementioned claims in the diagnosis, prognosis, risk assessment and/or prediction of a neoplastic disease is provided.
• the inventors have provided evidence that the presence of an enzyme and/or a metabolite of the kynurenine pathway in a given sample has a diagnostic, prognostic and/or predictive content with regard to neoplastic diseases.
• diagnostic methods like Immunohistochemistry (IHC) or standard ELISA (Enzyme linked Immuno Assay) or advanced EIA (Enzyme Immuno Assay, a technology using a tracer compound) or other immunohistochemical or immunodiagnostical methods.
• IHC Immunohistochemistry
• ELISA Enzyme linked Immuno Assay
• advanced EIA Enzyme Immuno Assay, a technology using a tracer compound
• said modulator and/or antagonist to an enzyme and/or a metabolite of the kynurenine pathway is labeled.
• Such label is, preferably, selected from the group consisting of a Radiolabel, Fluorescent label, a Luminescent label and/or an enzyme label.
• Said labeling can be direct, i.e., the antagonist itself is labeled.
• the labeling can be indirect (e.g., by means of a labeled secondary antibody which detectd the antagonist antibody).
• an enzyme and/or a metabolite of the kynurenine pathway for the development of a modulator and/or an antagonist against said enzyme and/or metabolite is provided, said modulator and/or antagonist being useful as a therapeutic, and/or a diagnostic agent.
• an antagonist against a novel, well described moiety be it a small molecule (like a metabolite) or a protein (like an enzyme) is within what the skilled person would consider as routine.
• the respective toolbox conjuggation to a carrier, immunization experiments, hybrodima technologies, affinity maturation, chimerization, humanization, display technologies, high throughput screening and the like is readily available to the skilled person.
• said metabolite of the kynurenine pathway is 3HAA, L-Kynurenine, Quinolinic Acid and/or Cinnabarinic Acid.
• said enzyme of the kynurenine pathway is Kynureninase.
• said modulator and/or antagonist is at least one modulator and/or antagonist according to the present invention.
• said development of a modulator and/or antagonist comprises at least one step of screening at least one library against the enzyme and/or a metabolite of the kynurenine pathway.
• a library can be an antibody library, e.g., as it is used for phage display or retrocyte display (see e.g. Hogenboom 2005).
• Such library can however also be a small molecule library, as e.g. described by Inglese et al (2007).
• a method of treatment of a neoplastic disease in a human or animal patient comprises the modulation of at least one parameter selected from the group consisting of the formation, concentration, availability and/or effect of 3HAA (3-Hydroxy Anthranilic Acid), L-Kynurenine, Quinolinic Acid and/or Cinnabarinic Acid, and/or Kynureninase.
• 3HAA 3-Hydroxy Anthranilic Acid
• L-Kynurenine L-Kynurenine
• Quinolinic Acid and/or Cinnabarinic Acid and/or Kynureninase.
• said method comprises the administration of a modulator and/or antagonist according to the present invention
• a method of diagnosis, prognosis, risk assessment and/or prediction of a physiological and/or pathological condition in which method the presence and/or concentration of an enzyme and/or a metabolite of the kynurenine pathway, either in free form and/or in form of the conjugated pool thereof, in a given sample is determined.
• conjugated pool relates to enzymes and/or a metabolites which are conjugated to other entities, i.e., to ubiquitin, to enzymes, to receptors or the like.
• the conjugated pool relates to any form of the target enzyme and/or metabolite which is not free.
• this can mean that in the conjugated pool the target is immunogenic enough to be detected by an antibody.
• the physiological and/or pathological condition is a neoplastic disease.
• the presence and/or concentration of an enzyme and/or a metabolite of the kynurenine pathway is determined in a tissue sample and/or in a liquid sample.
• Said tissue sample is for example a tissue slice, or a homogenized sample from a biopsy.
• Said liquid sample is for example a urine sample, saliva sample, blood serum sample, blood plasma sample, feces sample, sweat sample, swab sample, smear sample, a cell culture supernatant or the like.
• a method of research or screening is provided, in which method the presence and/or concentration of an enzyme and/or a metabolite of the kynurenine pathway, either in free form and/or in form of the conjugated pool thereof, in a given sample is determined.
• the term research relates to basic, fundamental or applied research.
• screening relates to methods in which large numbers of samples are screened, either for research purposes or for diagnosis or epidemiology.
• the presence and/or concentration an enzyme and/or a metabolite of the kynurenine pathway, either in free form and/or in form of the conjugated pool thereof, in a tissue sample and/or in a liquid sample is determined by at least one method selected from the group of
• realTime PCR also called rT PCR, or quantitative PCR
• rT PCR quantitative PCR
• Immunohistochemistry, ELISA, EIA and Immunofluorescence can be carried out on liquid samples, smears, biopsies, sections of tissue blocks, tissue microarrays.
• the quantification of the detected analytes can be carried out by using, e.g., microscopy, laser scanning cytometry or flow cytometry.
• Immunohistochemistry, ELISA, EIA and Immunofluorescence can be used to detect enzymes and metabolites of the kynurenine pathway.
• the presence and/or concentration of said enzyme and/or metabolite of the kynurenine pathway in a tissue sample and/or in a liquid sample is determined by at using at least one modulator and/or antagonist according to the present invention according.
• the enzyme and/or the metabolite of the kynurenine pathway is at least one selected from the group consisting of 3HAA, (3-Hydroxy Anthranilic Acid), L-Kynurenine, Quinolinic Acid, Cinnabarinic Acid, and/or Kynureninase.
• a physiological and/or pathological condition is (i) diagnosed, (ii) prognosed, (iii) it's risk is assessed, (iv) a prediction is made, wherein such condition is characterized by any form of activity of Indoleamine 2,3-dioxygenase l(IDOl), and/or Indoleamine 2,3 dioxygenase 2 (ID02), and/or Tryptophan 2,3-dioxygenase (TD02).
• the sample is characterized by any form of activity of Indoleamine 2,3-dioxygenase l(IDOl), and/or Indoleamine 2,3 dioxygenase 2 (ID02), and/or Tryptophan 2,3-dioxygenase (TD02).
• the term "characterized by any form of activity of IDOl, ID02 and/or TD02" relates to conditions in which the latter enzymes exert an activity which may lead to a pathological condition. This may be, for example, caused by overexpression, or by expression of a modified mutant, of IDOl, ID02 and/or TD02.
• the following section describes, in contrast thereto, the production of an antibody against a non-protein target, i.e., the small molecule 3HAA.
• the inventors have carried out such experiments also to create antibodies against other metabolites of the Kynurenine pathway, in particular against L-Kynurenine, Quinolinic Acid and/or Cinnabarinic Acid, but to avoid repetitions only the production of anti 3HAA antibodies is described in the following.
• 3HAA was conjugated to bovine serum albumine (BSA) by means of a carbodiimide crosslmker, which reacts with the carboxylic function of 3HAA and free amine functions of the BSA, to form a stable amide bond.
• BSA bovine serum albumine
• the "Imject BSA and EDC Conjugation Kit” provided by Thermo Scientific was used for this purpose.
• Monoclonal antibodies were generated following the established method of Kohler and Milstein (Kohler & Milstein 1975). Briefly, lymphocytes were isolated from mice immunized three times with the 3HAA-BSA conjugates. The lymphocytes were then fused with murine myeloma cells (SP20-Ag) with polyethyleneglycol (PEG 1500) to obtain hybridoma cells. The selection of hybridomas cells was realized by enzyme-linked immunosorbent assay (ELISA).
• ELISA enzyme-linked immunosorbent assay
• Clone 5B2-G2 was selected as the most promising clone.
• Monoclonal antibodies to 3HAA produced by Clone 5B2-G2 had an affinity of 10 "10 M (calculated based on the conjugates amount used to make the competition assay in ELISA, wherein the amount of conjugates is related to amount of BSA). No cross reactions with other metabolites from the kynurenines pathway could be found.
• the Clone 1B10 hybridoma cells have been deposited on May 31, 2012 at the "Collection Nationale de Culture de Microorganismes" (CNCM) at Pasteur Institute (25 rue du Dondel Roux F-75724 PARIS Cedex 15). The deposit has been made by means of 12 cryotubes containing more than 1 * 10 6 units as specified. The deposit number is CNCM 1-4637.
• EIA Enzyme Immunoassay
• EIA is different from the classic ELISA since a tracer is used.
• HRP Horseradish peroxidase
• 3D4-F2 antibodies were previously incubated at 0,01mg/ml with increasing concentration of the respective conjugate in EIA buffer for 1 hour at 37°C. Then, a tracer was added to the solution at a final concentration of ⁇ g/ml. Four washes in PBS-T were performed and revelation acetate buffer plus tetratmethylbenzidine allowed antibodies detection. Optical density (OD) was evaluated at 450 nm. Results were represented by the ratio B/BO, where BO is the OD obtained with 3D4-F2 and the Kynurenine-HRP alone (without the conjugate) and B the OD obtained at a specific concentration. The affinity was evaluated at 10 "9 M.
• a derivatization step is necessary to make the molecule reacting with amine containing molecules (eg proteins).
• amine containing molecules eg proteins
• a biological fluid eg sera
• EDC carbodiimide
• NHS N-hydroxysuccinimide
• the HRP -Kynurenine was then added at ⁇ g/ml and the antibodies solution containing the derivatization product plus the tracer was incubated for lh30 on the plate.
• Four washes in PBS-T were performed and revelation acetate buffer plus tetratmethylbenzidine allowed antibodies detection.
• Optical density (OD) was evaluated at 450 nm. Results were represented by the ratio B/B0 where B0 is the OD obtained with the 3D4-F2 and the Kynurenine-HRP alone (without derivatization product) and B the OD obtained at a specific concentration.
• the affinity for the free L-Kynurenine was estimated at 5* 10 ⁇ 6 M.
• HRP Horseradish peroxidase
• Solutions containing different amount of 3HAA were prepared in Dimethylsulfoxide supplemented with triethylamine (TEA) to increase the pH. Then, the derivatization process was performed using ethylchloro formate (ECF) diluted in Dimethylformamide (DMF) for 10 minutes at room temperature. Then, the reaction product was added drops by drops to 2 mg of BSA diluted in alkaline water (using TEA). This final solution was incubated with the 5B2- G2 antibodies at 0,01 mg/ml for lh at 37°C. The HRP-3HAA was then added at ⁇ g/ml and the antibodies solution containing the derivatization product plus the tracer was incubated for lh30 on the plate.
• ECF ethylchloro formate
• DMF Dimethylformamide
• HRP Horseradish peroxidase
• Dako antibody diluent
• BSA Sigma- Aldrich
• Anti L-Kynurenine mAb (3D4-F2) was then added at 0,01mg/ml, in the presence of 2% of normal goat serum, and incubated overnight at 4°C. Sections were washed three times in TBS, and incubated for 30 minutes with envision system (dextran polymer grafted with anti mouse IgG conjugated with HRP, Dako) at room temperature. Sections were washed three times before revelation with DAB (Dako) for 10 minutes at room temperature.
• Sections were rinsed, subjected to hematoxylin, dehydrated and mounted in DPX mountant media (Sigma-Aldrich). Pictures were obtained after a systematic scan of all cores (TissueGno sites). Quantification was performed according to the following grades:
• T054a The same human colon tumours Tissue Micro Array (T054a) was used to evaluate the 3HAA production.
• Dako antibody diluent
• BSA Sigma- Aldrich
• Anti 3HAA mAb (5B2-G2) was then added at 0,001mg/ml, in the presence of 2% of normal goat serum, and incubated overnight at 4°C. Sections were washed three times in TBS, and incubated for 30 minutes with envision system (dextran polymer grafted with anti mouse IgG conjugated with HRP, Dako) at room temperature. Sections were washed three times before revelation with DAB (Dako) for 10 minutes at room temperature. Sections were rinsed, subjected to hematoxylin, dehydrated and mounted in DPX mountant media (Sigma-Aldrich). Pictures were obtained after a systematic scan of all cores (TissueGno sites).
• 3HAA a mouse model of intracerebral glioblastoma. This model was obtained by intracerebral injection of GL261 to immunocompetent mice. After 29 days, brains were taken, and prepared for immunohistochemistry. In these samples, 3HAA was detectable in tumour cells but also in reactive surrounding astrocytes (see Fig. 15)
• T054a The same human colon tumours Tissue Micro Array (T054a) was used to evaluate the CA production.
• Dako antibody diluent
• BSA Sigma- Aldrich
• Anti Cinnabarinic Acid mAb (5C5-E10) was then added at 0,05 mg/ml, in the presence of 2% of normal goat serum, and incubated overnight at 4°C. Sections were washed three times in TBS, and incubated for 30 minutes with envision system (dextran polymer grafted with anti mouse IgG conjugated with HRP, Dako) at room temperature. Sections were washed three times before revelation with DAB (Dako) for 10 minutes at room temperature. Sections were rinsed, subjected to hematoxylin, dehydrated and mounted in DPX mountant media (Sigma- Aldrich). Pictures were obtained after a systematic scan of all cores (TissueGnostics).
• helper T cells activated with CD3/CD28 cocktail antibodies.
• Helper T cells were incubated with or without 3HAA and stained with CFSE (carboxyfluorescein succinimidyl ester).
• CFSE carboxyfluorescein succinimidyl ester
• HT29 ATCC - HTB38
• HCT116 ATCC - CCL247
• cells were plated on a 24 well/plate at 5* 104 cells/wells for 24hours. Cells were then incubated with a culture supernatant of our 7C7-A2 hybridoma for 48 hours. A blind cell-count was then performed using a Malassez cell.
• the 3HAA antibody was administered six days after cell injection (50 ⁇ g/mice) and was repeated once weekly with the same amount. A significant decrease of tumor growth was observed (Fig. 16).
• glioblastoma is an aggressive tumor without an adequate therapeutic solution
• the 3HAA antibody possesses therefore significant anti-tumor properties when administered subcutaneously at low amounts.
• GL261 (5* 10 7 /mL) were inoculated in 2 ⁇ iL of saline supplemented with 4% FCS stereotactically (David Kopf Instruments) using a 10 ⁇ _, Hamilton syringe.
• mice treated with the vehicle survived while the % of survival protection was 12,5% for IgG anti 3HAA (1B10), 25% for IgG anti 3HAA (5B2-G2), 12,5% for IgG anti L-Kynurenine (3D4-F2) and 12,5% for IgG anti Cinnabarinic Acid (5C5-E10).
• murine monoclonal antibodies in clinical settings is limited by the human anti- murine antibodies (HAMA) response against both variable and constant regions of the the murine Abs (Reynolds et al. 1989).
• murine antibodies can be humanized.
• the first technology to addresss this need was the grafting of the complementary determining residues (CDRs) onto the variable light (VL) and variable heavy (VH) frameworks of human immunoglobulin molecules (Winter et al 1993).
• VL variable light
• VH variable heavy
• the remaining mice CDRs of the humanized antibodies can still generate an anti-idiotypic (anti- Id) response in patients.
• SDRs specificity determining residues
• the most important CDR residues in the antibody-ligand binding are thus grafted.
• the last method is used to generate a humanized anti 3HAA antibody.
• the humanized anti 3HAA antibody is able to recognize the conjugated 3HAA with the same affinity as the murine anti 3HAA antibody, and is still specific enough when tested against other metabolites of the kynurenine pathway.
• a transgenic mouse (HuMab TM mouse debveloped by medarex) is used.
• the endologoues immunoglobulin gene repertoire has been replaced by its human counterpart, so that, after immunization, said mouse produces fully-human
• irradiated BALB/c Rag2-/-IL-2Ryc-/- mice reconstituted with human hematopoietic progenitor cells (hHPC) (Shultz et al 2007) are used.
• mice are immunized with 50 ⁇ g of 3HAA/BSA conjugates solubilised in ⁇ in NaCl 9g/l and emulsified with ⁇ of Freund complete ajuvant (1st immunization) and in Freund incomplete adjuvant, for the 2nd and 3rd immunizations. Immunizations are repeated every 2 weeks. The mice are sacrificed 2 weeks after the 3 rd immunization and the spleens recovered and splenocytes are isolated. The latter are then fused with a human myeloma cell (Karpas 707, see Karpas et al. 2001) line to generate hybridoma cell lines.
• a human myeloma cell Kerpas 707, see Karpas et al. 2001
• Methods to produce a human antibody against a hapten or self antigen by means of phage display are, e.g., discussed in Brichta et al (2005), Kerrm et al (2003), Keith et al. (2001) or Sheedy et al (2007).
• 3HAA is conjugated to both keyhole limpet haemocyanin (KLH) and bovine serum albumin (BSA) via linkage to 2-mercaptoethylamine, and hapten load/carrier protein is determined to be between eight and 10 haptens per BSA molecule using matrix assisted laser desorption spectrometry.
• KLH keyhole limpet haemocyanin
• BSA bovine serum albumin
• the Griffin library (MRC Laboratories, Cambridge, UK) consists of the majority of human VH and VL chain gene segments used in vivo, with CDR3 diversity generated using synthetic oligonucleotides (semi- synthetic).
• the Tomlinson library (MRC Laboratories, Cambridge, UK) is based on a single human framework with side chain diversity (DVT encoded) incorporated at 18 amino Acid positions in the antigen binding site (synthetic).
• the antibodies are displayed as scFv fragments on the coat protein of filamentous bacteria in the phagemid vector pHEN II.
• the phagemid clones are maintained and propagated in T-phage resistant E. coli TGlTr (Stratagene).
• Antibody fragments are expressed using the dicistronic, expression vector pIMS147.
• the vector is inducible with isopropyl L-D-thiogalactosidase (IPTG) and downstream from the scFv genes contains a human CU domain (forming a single chain antibody or scAb) for immunodetection and a hexahistidine tail for purification by nickel chelate affinity chromatography.
• the antibody expression vector pIMS147 is maintained in E. coli strain XL- 1 Blue (Stratagene).
• One hundred micro litres of either Tomlinson or Griffin glycerol stock are inoculated into 100 ml 2xTY broth containing 1 % glucose and 100 ⁇ g ml "1 ampicillin (2xTY-glu-amp), and incubated with shaking at 37°C to an OD 600 of 0.4.
• KM13 helper phage (2xlO n pfu) are added to 50 ml of each library culture and the mixture incubated at 37°C without shaking for 30 min.
• Infected cells are pelleted, resuspended in 100 ml 2xTY broth-0.1 % glu-amp-50 ⁇ g ml "1 kanamycin, and incubated overnight with shaking at 30°C. Phage particles are concentrated from each culture supernatant by precipitation with 20 ml polyethylene glycol in 2.5 M NaCl (20% w/v) as described previously.
• Two immunotubes are coated overnight with 100 ⁇ g ml "1 3HAA-BSA in phosphate buffered saline (PBS), washed with PBS and blocked with 2% skimmed milk-PBS at room temperature for 2 h.
• the concentrated phage particles (approximately lxl 0 13 ) from each library (Griffin or Tomlinson) are added to the immunotubes.
• Specific scFv phage bound to the antigen, and the unbound phages are removed by washing.
• the bound scFv phage are eluted from the immunotube with triethylamine (TEA) and infected into exponential phase TGI cell culture suspension in 2xTY broth for 30 min before being pelleted and plated onto agar plates of TYE-glu-amp and incubated at 30°C overnight.
• the colonies are scraped into 5 ml of 2xTY- glu-amp-15% glycerol and stored at -80°C.
• Fifty micro litres of this stock are used to inoculate 50 ml fresh 2xTY-glu-amp and phage grown, infected and rescued as described above. Selection is repeated a further two times with the following modifications: pan 2, 3HAA -KLH (100 ⁇ g ml "1 ); pan 3, 3HAA-BSA (1 ⁇ g ml "1 ).
• Phage antibody clones that only recognise 3HAA conjugates and not BSA or KLH alone are further characterised using a monoclonal binding ELISA where the phage antibodies are added to the plate in the presence or absence of free 3HAA.
• phAbs showing reduction of binding compared with phAbs added to the plate alone are sequenced in both directions on an ABI377 automated DNA sequencer (P.E. Applied Biosystems, Foster City, CA, USA). DNA from clones found to have different H or L chain sequences are digested with Ncol and Notl, and the scFv genes cloned into the similarly digested soluble expression vector pIMS147 before transformation into electrocompetent E. coli XL-1 Blue.
• Antibodies are then identified which showed binding to 3HAA.
• Single E. coli XL-1 Blue colonies containing antibodies specific for 3HAA are grown overnight in 5 ml LB containing 1 % (w/v) glucose, 50 ⁇ g ml "1 amp and 12.5 ⁇ g ml "1 tetracycline at 37°C using published methods.
• Each culture is used to inoculate 50 ml Terrific broth (TB)-glu-amp-tet in 250-ml baffled flasks, and the culture is grown to an OD of 15.
• the cells are pelleted and resuspended in 50 ml fresh TB-amp before induction of antibody expression with IPTG (1 mM final concentration) for 4 h.
• the cells are pelleted, osmotically shocked and the supernatant containing the periplasmic fraction harvested, ready for purification.
• Kynureninase or L-Kynurenine hydrolase, is a pyridoxal phosphate dependent enzyme that catalyses the formation of (i) Kynurenine into Anthranilic acid, and (ii) 3HAA from 3- Hydroxy Kynurenine.
• Humans express one kynureninase enzyme that is encoded by the KYNU gene located on chromosome 2.
• Kynureninase belongs to the class V group of aspartate aminotransferase superfamily of structurally homologous pyridoxal 5'-phosphate (PLP) dependent enzymes.
• kynureninase has the ENZYME entry No EC 3.7.1.3. It has 465 AA residues and a molecular weight of about 52,4 kDa.
• Antibodies against kynureninase can be produced with the established method portfolio known to the skilled person, e.g., by mere immunization of a mammal (polyclonal antibodies), the Kohler Milstein technique (Mouse monoclonal antibodies), Chimerization (Chimeric recombinant antibodies), CDR grafting (humanized antibodies), Affinity maturation and DNA shuffling, Phage display from a human library (human antibodies), Transgenic mammal techniqures (human antibodies). See Lonberg 2005 for further reference.
• the presence of an enzyme and/or a metabolite of the kynurenine pathway may be useful in the diagnosis, prognosis, risk assessment and/or prediction of a neoplastic disease. This approach has been demonstrated by immunohistochemical detection of 3HAA in tumor tissue.
• 3HAA is a metabolite of the kynurenine pathway
• enzymes of the kynurenine pathway can of course likewise be detected by means of immunohistochemistry.
• proteins, and particularly enzymes can also be detected with modern molecular techniques, e.g., by detecting their mRNA in situ, or in a liquid sample.
• both approaches will be described for a selected enzyme of the kynurenine pathway, i.e.. kynureninase. It is important to understand that this approach will also be applicable to other enzymes of the kynurenine pathway.
• PBMC peripheral blood mononuclear cells
• lysis buffer e.g., Trizol
• RNA is then extracted (follow classic protocol).
• reverse transcriptase Qiagen
• real time PCR also termed quantitative PCR
• kynureninase expression levels takes place according to standard real time PCR protocols, e.g. with the FastLane Cell SYBR Green Kit provided by Qiagen. Generela technical infotrmation can furthermore be found in Logan & Saunders (2009).
• the Kynureninase expression level is compared to an house-keeping gene such as GAPFH to allow relative quantification. This method can be used to compare kynureninase level in physiologic vs pathologic situations.
• ISH In situ hybridization
• sequence specific probes have to be synthezised which are able to hybridize with a nucleic acid encoding for kynureninase (either genomic DNA, mRNA or cDNA). Said probes may serve as primers of PCR and/or as labelling probes.
• Suitable protocols for in situ hybridization can be found in Braissant O, Wahli W (1998), and suitable rotocols for in situ PCR can be found, e.g., in Nuovo (1995)
• Enzymes and metabolites of the kynurenine pathway can furthermore be detected with HPLC (high performance liquid chromatography), GC-MS (gas chromatography/mass spectrometry), and/or LC-MS (liquid chromatography/mass spectrometry). These methods are all within the scope of routine of the skilled person. Short description of the Figures
• Fig. 1 Immunochemical characterization of monoclonal antibodies directed against carrier conjugated L-Kynurenine.
• Fig. 2 Detection of both conjugated and free Kynurenine in a biological fluid using EIA (Enzyme immunoassay).
• Fig. 3 Immunochemical characterization of monoclonal antibodies directed against carrier conjugated 3HAA.
• Affinity comparison of the different monoclonal antibodies was performed with an ELISA competition assay and shows a higher affinity for the 5B2-G2 and 6F6-A2.
• B) Specificity of the 5B2-G2 antibody was evaluated with a competition assay and shown no cross-reaction with the anthranilic conjugate while 3HAA conjugate was recognized at an affinity around 10 "10 M.
• Fig. 4 Detection of both conjugated and free 3HAA in a biological fluid using EIA.
• Fig. 5 Immunochemical characterization of monoclonal antibodies directed against carrier conjugated Cinnabarinic Acid.
• Fig. 6 Detection of both conjugated and free Cinnabarinic Acid in a biological fluid using EIA.
• Fig. 7 Immunochemical characterization of monoclonal antibodies directed against carrier conjugated Quinolinic acid.
• Affinity comparison of the different monoclonal antibodies was performed with an ELISA competition assay and shows a higher affinity for the 3B2-C7.
• Fig. 8 L-Kynurenine detection in human colon carcinomas
• Fig. 10 3HAA detection in human colon carcinomas
• 3HAA presence was evaluated by IHC in 10 colon carcinomas and 2 normal colons on a tissue micro array section obtained from US Biomax (USA). An intermediate staining was observed in a healthy colon (Healthy) while Case 1 shows absence of 3HAA and Case 2 shows an intermediate 3HAA production/accumulation in the cytoplasm of tumour cells.
• Fig. 11 3HAA detection in human breast carcinomas
• 3HAA presence was evaluated by IHC in 10 breast carcinomas and 2 normal colons on a tissue micro array section obtained from US Biomax (USA). An intermediate staining was observed in an healthy breast epithelium (Healthy) while Case 1 shows weak production of 3HAA and Case 2 shows a strong 3HAA production/accumulation in the cytoplasm of tumour cells and cells from the tumoural stroma.
• Fig. 12 Cinnabarinic detection in human colon carcinomas
• Cinnabarinic Acid (CA) presence was evaluated by IHC in 10 colon carcinomas and 2 normal colons on a tissue micro array section obtained from US Biomax (USA). No staining was observed in an healthy colon (Healthy) while Case 1 shows a weak production of CA and Case 2 shows a strong CA production/accumulation in the cytoplasm of tumour cells.
• Fig. 13 Cinnabarinic Acid detection in human breast carcinomas
• CA presence was evaluated by IHC in 10 breast carcinomas and 2 normal colons on a tissue micro array section obtained from US Bio max (USA). An intermediate staining was observed in a healthy breast epithelium (Healthy) while Case 1 shows absence of CA and Case 2 shows a strong CA production/accumulation in the cytoplasm of tumour cells and cells from the tumoral stroma.
• Fig. 14 Benefits of 3HAA monoclonal antibodies in a mouse model of melanoma induced by B16-F10 tumour cells implantation into C57BL/6 mice.
• IgG anti 3HAA 1B10
• dacarbazine 100 ⁇ g of IgG anti 3HAA were administered subcutaneously at day 6 (at a time when tumours were detectable), 13 and 20.
• dacarbazine was administered intra-peritonealy at 80mg/kg. The treatment started at day 6 and was repeated once every 2 weeks for 4 consecutive times; chemotherapy cycles were repeated every 4 days.
• Fig. 15 Benefits of 3HAA monoclonal antibodies in a mouse model of Glioblastoma induced by GL261 tumour cells implantation into C57BL/6 mice.
• Fig. 16 Benefits of 3HAA monoclonal antibodies in a mouse model of Glioblastoma induced by GL261 tumour cells implantation into C57BL/6 mice.
• Fig. 17 Benefits of 3HAA monoclonal antibodies in an intracerebral model of glioblastoma obtained by GL261 tumour cells implantation into C57BL/6 mice.
• 3HAA Detection of 3HAA in tumour sections obtained from mice implanted intracerebrally with GL261. 3HAA was both detected in surrounding reactive astrocytes and tumour cells with a likely polarized localization.
• mice were treated 6 days after intracerebral GL261 implantation with 10( ⁇ g of IgG anti 3HAA (1B10) injected subcutaneously and was repeated once a weak. 29 Days after cell implantation, brains were taken, systematically sliced and tumour area photographed and measured. When compared to vehicle treated mice, IgG anti 3HAA reduced significantly the tumour volume.
• Fig. 18 Benefits of monoclonal antibodies directed against either L-Kynurenine (3D4-F2), 3HAA (1B10 and 5B2-G2) and Cinnabarinic Acid (5C5-E10) in an intracerebral model of Glioblastoma induced by GL261 tumour cells implantation into C57BL/6 mice.
• the step is catalyzed by either a) Indoleamine 2,3- dioxygenase (IDOl) or b) Tryptophan 2,3-dioxygenase (TD02). If one of the two is blocked, the reaction can still take place, while blocking both may have severe side effects.
• IDOl Indoleamine 2,3- dioxygenase
• TD02 Tryptophan 2,3-dioxygenase
• Fig. 20 Overview of the kynurenine pathway with its enzymes and metabolites.
• the enzymes are as follows: i) Kynurenine formamidase, a) Kynurenine amino-transferase, b) Kynurenine 3-hydroxylase (also called Kynurenine mono-oxygenase), c) Kynureninase (also called L- Kynurenine hydrolase), d) Kynurenine amino-transferase, e) Kynureninase (also called L- Kynurenine hydrolase), and f) 3-Hydroxyanthranilic Acid oxygenase (also called 3- Hydroxanthranilate dioxygenase).
• the metabolites are as follows: L-Formylkynurenine, Kynuramine, L-Kynurenine, Kynurenic Acid, 3-hydroxyL-kynurenine, Anthranilic Acid, 3-hydroxyanthranilic Acid, Xanthurenic Acid, Quinaldic Acid, Picolinioc Acid and/or Quinolinic Acid
• Fig. 21 Suppression of anti-pro liferative properties of 3HAA on helper T cells by a 3HAA monoclonal antibody (1B10).
• CD4 positive T cells were activated by anti CD3/CD28 antibodies cocktail and incubated for 96 hours with either vehicle, 3HAA at ⁇ or 3HAA ( ⁇ ⁇ ) + IgG anti 3HAA.
• 3HAA 3HAA monoclonal antibody
• anergy of T cells was induced by 3HAA, a phenomenon largely ablated when co-incubated with the 3HAA monoclonal antibody.
• Fig. 22 Anti-pro liferative effect of IgG anti Cinnabarinic acid on human colon cancer cell lines.
• HT29 and HCT116 cells were plated for 24 hours and incubated with 7C7-A2 culture supernatant for 48 hours. Cells were then counted and show a significant decrease of proliferation rate in HT29 while only a trend in the same way was observed in HCT116. References mentioned in the text
• RTPrimerDB the Real- Time PCR primer and probe database. Nucleic Acids Research, 31(1): 122-123.
• Prendergast GC Immune escape as a fundamental trait of cancer: focus on IDO. Oncogene 2008;27:3889-900.

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